Abstract
Membrane fusion during exocytosis and throughout the cell is believed to involve members of the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) family of proteins. The assembly of these proteins into a four-helix bundle may be part of the driving force for bilayer fusion. Regulated exocytosis in neurons and related cell types is specialized to be fast and Ca(2+)-dependent suggesting the involvement of other regulatory proteins specific for regulated exocytosis. Among these are the complexins, two closely related proteins that bind only to the assembled SNARE complex. We have investigated the function of complexin by analysis of single vesicle release events in adrenal chromaffin cells using carbon fiber amperometry. These cells express complexin II, and overexpression of this protein modified the kinetics of vesicle release events so that their time course was shortened. This effect depended on complexin interaction with the SNARE complex as introduction of a mutation of Arg-59, a residue that interacts with synaptobrevin in the SNARE complex, abolished its effects. The data are consistent with a function for complexin in stabilizing an intermediate of the SNARE complex to allow kiss-and-run recycling of the exocytosed vesicle.
Highlights
The SNARE proteins associate to form a SNARE complex (1) that has a crucial role in membrane fusion events throughout the secretory pathway as the core of a highly conserved fusion machinery (2)
We have investigated the function of complexin by analysis of single vesicle release events in adrenal chromaffin cells using amperometry and demonstrated a novel effect of complexin II on release kinetics that depends on its interaction with the SNARE complex
Our data show that overexpression of complexin II in chromaffin cells has two effects, an inhibition of the number of exocytotic events and changes in the kinetics of single vesicle release events consistent with kiss-and-run exocytosis (22)
Summary
The SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) proteins associate to form a SNARE complex (1) that has a crucial role in membrane fusion events throughout the secretory pathway as the core of a highly conserved fusion machinery (2). The SNARE proteins involved in regulated exocytosis in neurons and neuroendocrine cells, syntaxin, SNAP-25 and VAMP (synaptobrevin) have been studied in most detail (3,4). The neuronal SNAREs are sufficient for membrane fusion when reconstituted in liposomes (5) but this occurs in a Ca2+-independent manner and with kinetics that are many orders of magnitude slower than exocytosis at the synapse suggesting an essential requirement for other proteins. A prime candidate for a protein that influences membrane fusion during regulated exocytosis is complexin (6,7) as it binds to the assembled SNARE complex (6,8,9). We have investigated the function of complexin by analysis of single vesicle release events in adrenal chromaffin cells using amperometry and demonstrated a novel effect of complexin II on release kinetics that depends on its interaction with the SNARE complex. The data are consistent with a function for complexin in controlling the closure of the fusion pore to elicit rapid kiss-and run recycling of the exocytosed vesicle
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